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Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD). The pathogenesis of HLHS is poorly understood and due to the likely oligogenic complexity of the disease, definitive HLHS-causing genes have not yet been identified. Postulating that impaired cardiomyocyte proliferation as a likely important contributing mechanism to HLHS pathogenesis, and we conducted a genome-wide siRNA screen to identify genes affecting proliferation of human iPSC-derived cardiomyocytes (hPSC-CMs). This yielded ribosomal protein (RP) genes as the most prominent class of effectors of CM proliferation. In parallel, whole genome sequencing and rare variant filtering of a cohort of 25 HLHS proband-parent trios with poor clinical outcome revealed enrichment of rare variants of RP genes. In addition, in another familial CHD case of HLHS proband we identified a rare, predicted-damaging promoter variant affectingRPS15Athat was shared between the proband and a distant relative with CHD. Functional testing with an integrated multi-model system approach reinforced the idea that RP genes are major regulators of cardiac growth and proliferation, thus potentially contributing to hypoplastic phenotype observed in HLHS patients. Cardiac knockdown (KD) of RP genes with promoter or coding variants (RPS15A, RPS17, RPL26L1, RPL39, RPS15) reduced proliferation in generic hPSC-CMs and caused malformed hearts, heart-loss or even lethality inDrosophila. In zebrafish, diminishedrps15afunction caused reduced CM numbers, heart looping defects, or weakened contractility, while reducedrps17orrpl39function caused reduced ventricular size or systolic atrial dysfunction, respectively. Importantly, genetic interactions betweenRPS15Aand core cardiac transcription factorsTBX5in CMs,Drosocross, pannierandtinmanin flies, andtbx5andnkx2-7(nkx2-5paralog) in fish, support a specific role for RP genes in heart development. Furthermore,RPS15AKD-induced heart/CM proliferation defects were significantly attenuated byp53KD in both hPSC-CMs and zebrafish, and by Hippo activation (YAP/yorkieoverexpression) in developing fly hearts. Based on these findings, we conclude that RP genes play critical roles in cardiogenesis and constitute an emerging novel class of gene candidates likely involved in HLHS and other CHDs.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We therefore performed whole genome sequencing (WGS) on a large cohort of HLHS patients and their families to identify candidate genes that were then tested in Drosophila heart model for functional and structural requirements. Bioinformatic analysis of WGS data from an index family comprised of a HLHS proband born to consanguineous parents and postulated to have a homozygous recessive disease etiology, prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of the candidate HLHS gene homologs tested, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. Interestingly, these heart defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex’s role in maintaining cristae morphology and ETC complex assembly. Analysis of 183 genomes of HLHS patient-parent trios revealed five additional HLHS probands with rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes in these cases for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (E3 ubiquitin ligase), or SPTBN1 (scaffolding protein) caused synergistic heart defects, suggesting the potential involvement of a diverse set of pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

Background: KCNMA1 encodes the α-subunit of the large-conductance Ca 2+ -activated K + channel, K Ca 1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of K Ca 1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. Methods: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of K Ca 1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. Results: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of K Ca 1.1 in normal hearts using immunostaining and immunogold electron microscopy. K Ca 1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the K Ca 1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the K Ca 1.1 ortholog, kcnma1b , in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila K Ca 1.1 ortholog, slo , systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo -deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the K Ca 1.1 loss-of-function models. Conclusions: Our data point to a highly conserved role of K Ca 1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that K Ca 1.1 loss of function may predispose to AF.

BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca(2+)-activated K(+) channel, K(Ca)1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of K(Ca)1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of K(Ca)1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of K(Ca)1.1 in normal hearts using immunostaining and immunogold electron microscopy. K(Ca)1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the K(Ca)1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the K(Ca)1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila K(Ca)1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the K(Ca)1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of K(Ca)1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that K(Ca)1.1 loss of function may predispose to AF.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developingDrosophilaand zebrafish hearts revealed that LDL receptor-related proteinLRP2is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to parents the proband’s iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.

Congenital heart diseases (CHD), such as hypoplastic left heart syndrome (HLHS), are considered to have complex genetic underpinnings that are poorly understood. Here, an integrated multi-disciplinary approach was applied to identify novel genes and underlying mechanisms associated with HLHS. A family-based strategy was employed that coupled whole genome sequencing (WGS) with RNA sequencing of patient-derived induced pluripotent stem cells (iPSCs) from a sporadic HLHS proband-parent trio to identify, prioritize and functionally evaluate candidate genes in model systems. Consistent with the hypoplastic phenotype, the proband’s iPSCs had reduced proliferation capacity. Filtering WGS for rare de novo, recessive, and loss-of-function variants revealed 10 candidate genes with recessive variants and altered expression compared to the parents’ iPSCs. siRNA/RNAi-mediated knockdown in generic human iPSC-derived cardiac progenitors and in thein vivo Drosophilaheart model revealed that LDL receptor related proteinLRP2and apolipoproteinAPOBare required for robust hiPSC-derived cardiomyocyte proliferation and normal hear structure and function, possibly involving an oligogenic mechanism via growth-promoting WNT and SHH signaling.LRP2was further validated as a CHD gene in a zebrafish heart model and rare variant burden testing in an HLHS cohort. Collectively, this cross-functional genetic approach to complex congenital heart disease revealed LRP2 dysfunction as a likely novel genetic driver of HLHS, and hereby established a scalable approach to decipher the oligogenic underpinnings of maladaptive left heart development.One sentence summaryWhole genome sequencing and a multi-model system candidate gene validation - human iPSC-derived cardiomyocytes andDrosophilaand zebrafish hearts - identified lipoproteinLRP2as a new potential driver in congenital heart disease and suggests a deficit in proliferation as a hallmark of hypoplastic left heart syndrome.